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1. Vorbereitung der Aptamer-Funktion Stock <ol><li> Folgen Sie den Anweisungen des Herstellers und lösen das Oligonukleotid (5-Cy5-CCTG GGGGAGTATTGCGGAGGAAGG-3, Sequenz aus Referenz 18) in Wasser, einer 100-uM Endkonzentration zu erreichen. </li><li> Bereiten Sie die Aptamer - Arbeitslösung durch Verdünnen des Oligonukleotid - Lager auf 200 nM mit Bindungspuffer (20 mM Tris, pH 7,6; 300 mM NaCl, 5 mM MgCl 2; 0,01% Tween 20). </li><li> Inkubieren Sie die Mischung für 2 min bei 90 ° C, lassen Sie sofort die Probe auf Eis abkühlen, und die Probe bei Raumtemperatur verwenden. </li></ol> 2. Herstellung der Ligand Verdünnungsreihe <ol><li> Für jeden Liganden (Adenosin-Triphosphat (ATP), Adenosindiphosphat (ADP), Adenosinmonophosphat (AMP), Adenin, Guanosin-Triphosphat (GTP), Cytosin-triphosphat (CTP), Desoxyadenosintriphosphat (dATP) und S-Adenosylmethionin (SAM) ; jeweils) 10 mM Lager, bereiten Sie einen 16-stufigen seriellen dilutiauf in 200 &amp; mgr; l Mikroreaktionsröhrchen. HINWEIS: Die Zentrifugation von Ligand Bestände für 5 min bei 14000 xg helfen können Aggregate zu entfernen. Geringes Volumen, geringe Bindungsreaktionsrohre werden empfohlen Adsorption von Molekülen an den Rohrwänden zu vermeiden. </li><li> Beginnen mit einer maximalen Konzentration von mindestens 50 mal höher ist als die geschätzte Affinität und Verringerung um 50% in jedem Verdünnungsschritt die Ligandenkonzentration. HINWEIS: Die Konzentration finder Werkzeug in die Steuerungssoftware implementiert simuliert Bindungsdaten und hilft bei der richtigen Konzentrationsbereich für die Verdünnungsreihe finden. </li><li> Füllen Sie 20 ul des Liganden Lager (10 mM) in Rohr 1. Fügen Sie 10 ul Aptamer-Bindungspuffer in Mikroreaktionsrohre 2 bis 16. </li><li> Transfer 10 ul Rohr 1 bis Rohr 2 und ordnungsgemäß durch Auf- und Abpipettieren mehrmals mischen. Übertragen Sie 10 &amp; mgr; l in die nächste Röhre und wiederholen Sie diese Verdünnung für die verbleibenden Rohre. </li><li> Entsorgen Sie die 10 ul Überschuss aus dem letzten Rohr. Vermeiden Sie bUffer Verwässerungseffekte. Der Puffer in Rohr 1 und in Röhrchen 2-16 müssen identisch sein. </li></ol> 3. Herstellung der endgültigen Reaktions Mix <ol><li> Bereiten Sie die einzelnen Bindungsreaktionen mit einem Volumen von 20 &amp; mgr; l (10 &amp; mgr; l Arbeitslösung Aptamer + 10 &amp; mgr; l des jeweiligen Liganden Verdünnung) Pipettierfehler zu minimieren. Ein Volumen von nur 4 ul reicht aus ll die Kapillare zu fi nden. </li><li> In 10 ul der 200 nM Aptamer-Arbeitslösung zu 10 &amp; mgr; l jedes Liganden Verdünnung und mischen richtig durch Auf- und Abpipettieren mehrmals. </li><li> Inkubiere die Proben für 5 min bei Raumtemperatur und fi ll die Proben in Standard-Kapillaren durch die Kapillaren in die Probe eingetaucht wird. Längere Inkubationszeiten können für einige Wechselwirkungen erforderlich sein; jedoch 5 min ist ausreichend für die meisten. Berühren die Kapillaren nur an den Seiten, nicht auf dem mittleren Teil, wo die optische Messung genommen wird. </li><li> Legen Sie die Kapillaren auf the Kapillar-Fach und die MST-Gerät starten. </li></ol> 4. Starten des MST-Gerät HINWEIS: Das Gerät verfügt über zwei vorinstallierte Software-Pakete, die &#39;&#39; control &quot;Software für den technischen Aufbau der experimentellen Bedingungen und der &#39;&#39; Analyse&quot; Software für die Interpretation der erzeugten Daten. <ol><li> Vor der Platzierung der Kapillare Fach in die MST-Gerät, starten Sie die Steuerungssoftware und stellen Sie die gesamte gewünschte Temperatur von &#39;Auswahl von&#39; ermöglichen Steuerung manueller Temperatur &quot;in der &#39;&#39; Temperaturregelung&quot; Drop-Down-Menü. Einstellen der Temperatur auf 25 ° C in dieser Weise. HINWEIS: Die MST Instrumente können von 22 bis 45 ° C temperiert werden. </li><li> Warten, bis die Temperatur das erwartete Niveau zu erreichen, und legen Sie dann die Kapillare Fach in die MST-Gerät. </li><li> Stellen Sie den LED-Kanal &#39;&#39; rot &quot;für Cy5 Farbstoffen und stellen Sie die LED-Leistung ein fl uorescenc zu gewinnene Signal von 300 bis 1000 Fluoreszenzeinheiten an der MST-Gerät mit einem Standard-Sensor. 25% LED-Leistung wird in dieser Studie verwendet. HINWEIS: 6.000 bis 18.000 Fluoreszenzeinheiten für die MST mit einem hochempfindlichen Sensor empfohlen. </li></ol> 5. Kapillar-Scan <ol><li> Führen Sie eine Kapillare Scan verschiedene Qualitätsaspekte der Probe zu überprüfen, indem die Kapillare Position auf der &quot;Control&quot; Software ein und klicken auf &quot;Start Kappe scan&quot; bevor die MST-Messung starten. </li><li> Überprüfen Sie die Kapillar-Scan für Fluoreszenzverstärkung / Lösch- und Klebeeffekte (U-förmige oder abgeflachte Spitzen) in der Software. HINWEIS: Weitere Informationen über die Erkennung und Behandlung von Fluoreszenz und Klebeeffekte können in der Diskussion zu finden. </li></ol> 6. MST Mess HINWEIS: Vor der Messung MST beginnen, stellen Sie sicher, dass Effekte, Verbesserung / Löscheffekte auszuschließen kleben, oderPipettierfehler, und sicherzustellen, dass die Kapillare Scan zeigt, dass die Fluoreszenz- Signal reicht aus. Weitere Details, die Diskussion. <ol><li> Ordnen die Ligandenkonzentrationen aus der Verdünnungsreihe auf die jeweilige Kapillare Position in der &#39;&#39; control &quot;Software Betrachten der Verdünnungsschritt das Aptamer und dem Liganden zu mischen. (1: 1). </li><li> Geben Sie die höchste Konzentration an Ligand (5 mM) für kapillare # 1, wählen Sie die richtige Verdünnung (hier 1: 1), klicken Sie auf die maximale Konzentration, und verwenden Sie die Drag-Funktion, um automatisch die verbleibenden Konzentrationen in Kapillaren zuweisen # 2- 16. Die niedrigste Konzentration ist 152,6 nM. </li><li> Geben Sie die Konzentration des Fluoreszenz- Aptamer (hier 100 nM) in dem jeweiligen Abschnitt der Steuerungssoftware. </li><li> Verwenden Sie die Standardeinstellungen, die die fluoreszenz für 5 Sekunden erkennen, notieren Sie die MST für 30 Sekunden, und notieren Sie die Fluoreszenz für weitere 5 Sekunden nach der Inaktivierung des Lasers t<html> o die Rückdiffusion von Molekülen zu überwachen. </li><li> Stellen Sie die Laserleistung auf 20% in dem jeweiligen Abschnitt der Steuersoftware. ANMERKUNG: Um das beste Signal-zu-Rausch-Verhältnis zu erhalten und zu unspezifischen Effekte zu vermeiden, wird eine Laserleistung von 20-40% empfohlen. In bestimmten Fällen kann eine höhere Laserleistung eine gute Trennung von ungebundenen und gebundenen Moleküle zu erhalten erforderlich. </li><li> Speichern Sie das Experiment nach den Zielordner auswählen und auf die MST Messung starten durch Drücken der &#39;&#39; Start MST Messung &quot;-Taste. HINWEIS: Die .ntp Datei im Zielordner erzeugt wird. Mit diesem Setup dauert eine Messung 10-15 min. </li><li> Wiederholen Sie die experimentellen Verfahren mindestens zweimal für eine genauere Bestimmung des Wertes 50 EC. HINWEIS: Um die technische Reproduzierbarkeit zu testen, können die gleichen Kapillaren mehrmals gescannt werden (technische Wiederholungen). </li></ol> 7. MST Datenanalyse nt &quot;&gt; Hinweis: Die Analyse - Software die Analyse von Daten über die fl y während der Messung ermöglicht die Analyse - Software , die MST Zeit Spuren und Veränderungen in der normierten Fluoreszenz- (F Norm) gegen die Ligandenkonzentration 37 aufgetragen ist .. <ol><li> Starten Sie die MST-Analyse-Software (MO.Affinity Analysis) und laden Sie die .ntp Datei aus dem Zielordner. Wählen Sie &quot;MST&quot; als Analysetyp in der Datenauswahlmenü. HINWEIS: Bei Liganden-abhängige Fluoreszenzeffekte kann die anfängliche Fluoreszenz zur Analyse ausgewählt werden. </li><li> Fügen Sie die entsprechenden technischen oder biologischen Lauf (s) einer neuen Analyse per Drag-and-Drop oder über die Schaltfläche &quot;+&quot; unterhalb der jeweiligen Versuchslauf drücken. </li><li> Drücken Sie die Informationstaste unterhalb der jeweiligen Versuchslauf zu erhalten Informationen über die Eigenschaften des Experiments MST Spuren, Kapillar-Scan, Kapillar-Form, anfängliche Fluoreszenz und Bleichrate. Hinweis: Diese Rohdaten können also in späteren Schritten der Analyse überprüft werden. </li><li> die MST Spuren für die Aggregation und Fällung Effekte sichtbar, wie Beulen und Spikes einer Sichtprüfung. HINWEIS: Weitere Informationen über die Erkennung und Behandlung von Aggregationseffekten, lesen Sie die Diskussion. </li><li> die kapillare Scan und die kapillare Form Overlay für Adsorptionseffekten, sichtbar als abgeflacht oder U-förmigen Spitzen Sichtprüfung. Eine Sichtprüfung des Kapillar-Scan und die anfängliche Fluoreszenz für Fluoreszenzeffekte. Sichtprüfung für Photobleaching Auswirkungen der Bleichrate. </li><li> Wechseln Sie auf die Dosis-Wirkungs-Modus und ändern Sie die Analyse-Einstellung auf &quot;Experten&quot; -Modus indem Sie die entsprechende Taste drücken. Wählen Sie &quot;T-Jump&quot;, wie die MST Bewertungsstrategie. </li><li> Wählen Sie den &quot;Hügel&quot; Modell für die Kurve Armatur. Die Bindungsparameter werden automatisch berechnet. Normalisieren der Daten durch die jeweilige Art der Normalisierung in der &quot;vergleichen Sie die Ergebnisse&quot; Menü wählen. exportieren Sie dieDaten entweder als .xls oder .pdf. HINWEIS: Die unten stehende Tabelle der Bindungs ​​Diagramm fasst die berechneten Bindungsparameter. </li></ol>

<ol><li>Linke, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Automated+Microscale+Thermophoresis+Screening+Approach+for+Fragment-Based+Lead+Discovery%5BTitle%5D)+AND+%22J+Biomol+Screen%22%5BJournal%5D)">An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery.</a> J Biomol Screen. 21 (4), 414-421 (2015).</li>
<li>Jerabek-Willemsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroScale+Thermophoresis%3A+Interaction+analysis+and+beyond%5BTitle%5D)+AND+%22Journal+of+Molecular+Structure%22%5BJournal%5D)">MicroScale Thermophoresis: Interaction analysis and beyond.</a> Journal of Molecular Structure. 1077, 101-113 (2014).</li>
<li>Zillner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscale+thermophoresis+as+a+sensitive+method+to+quantify+protein%3A+nucleic+acid+interactions+in+solution%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution.</a> Methods Mol Biol. 815, 241-252 (2012).</li>
<li>Zhang, W., Duhr, S., Baaske, P., Laue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscale+thermophoresis+for+the+assessment+of+nuclear+protein-binding+affinities%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Microscale thermophoresis for the assessment of nuclear protein-binding affinities.</a> Methods Mol Biol. 1094, 269-276 (2014).</li>
<li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nat Commun. 1, 100(2010).</li>
<li>Seidel, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+microscale+thermophoresis+discriminates+sites+and+affinity+of+protein-ligand+binding%5BTitle%5D)+AND+%22Angew+Chem+Int+Ed+Engl%22%5BJournal%5D)">Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.</a> Angew Chem Int Ed Engl. 51 (42), 10656-10659 (2012).</li>
<li>Seidel, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscale+thermophoresis+quantifies+biomolecular+interactions+under+previously+challenging+conditions%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.</a> Methods. 59 (3), 301-315 (2013).</li>
<li>Schubert, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Df31+protein+and+snoRNAs+maintain+accessible+higher-order+structures+of+chromatin%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Df31 protein and snoRNAs maintain accessible higher-order structures of chromatin.</a> Mol Cell. 48 (3), 434-444 (2012).</li>
<li>McKeague, M., Derosa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+and+opportunities+for+small+molecule+aptamer+development%5BTitle%5D)+AND+%22J+Nucleic+Acids%22%5BJournal%5D)">Challenges and opportunities for small molecule aptamer development.</a> J Nucleic Acids. 2012, 748913(2012).</li>
<li>Ruscito, A., DeRosa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-Molecule+Binding+Aptamers%3A+Selection+Strategies%2C+Characterization%2C+and+Applications%5BTitle%5D)+AND+%22Front+Chem%22%5BJournal%5D)">Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications.</a> Front Chem. 4, 14(2016).</li>
<li>Chang, A. L., McKeague, M., Liang, J. C., Smolke, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+and+equilibrium+binding+characterization+of+aptamers+to+small+molecules+using+a+label-free%2C+sensitive%2C+and+scalable+platform%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform.</a> Anal Chem. 86 (7), 3273-3278 (2014).</li>
<li>Chang, A. L., McKeague, M., Smolke, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facile+characterization+of+aptamer+kinetic+and+equilibrium+binding+properties+using+surface+plasmon+resonance%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance.</a> Methods Enzymol. 549, 451-466 (2014).</li>
<li>Jing, M., Bowser, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+measuring+aptamer-protein+equilibria%3A+a+review%5BTitle%5D)+AND+%22Anal+Chim+Acta%22%5BJournal%5D)">Methods for measuring aptamer-protein equilibria: a review.</a> Anal Chim Acta. 686 (1-2), 9-18 (2011).</li>
<li>Sokoloski, J. E., Dombrowski, S. E., Bevilacqua, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermodynamics+of+ligand+binding+to+a+heterogeneous+RNA+population+in+the+malachite+green+aptamer%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Thermodynamics of ligand binding to a heterogeneous RNA population in the malachite green aptamer.</a> Biochemistry. 51 (1), 565-572 (2012).</li>
<li>Burnouf, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((kinITC%3A+a+new+method+for+obtaining+joint+thermodynamic+and+kinetic+data+by+isothermal+titration+calorimetry%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry.</a> J Am Chem Soc. 134 (1), 559-565 (2012).</li>
<li>Mannironi, C., Scerch, C., Fruscoloni, P., Tocchini-Valentini, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+recognition+of+amino+acids+by+RNA+aptamers%3A+the+evolution+into+an+L-tyrosine+binder+of+a+dopamine-binding+RNA+motif%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif.</a> RNA. 6 (4), 520-527 (2000).</li>
<li>Jenison, R. D., Gill, S. C., Pardi, A., Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+molecular+discrimination+by+RNA%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">High-resolution molecular discrimination by RNA.</a> Science. 263 (5152), 1425-1429 (1994).</li>
<li>Huizenga, D. E., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+DNA+aptamer+that+binds+adenosine+and+ATP%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">A DNA aptamer that binds adenosine and ATP.</a> Biochemistry. 34 (2), 656-665 (1995).</li>
<li>Lee, E. R., Baker, J. L., Weinberg, Z., Sudarsan, N., Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+allosteric+self-splicing+ribozyme+triggered+by+a+bacterial+second+messenger%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">An allosteric self-splicing ribozyme triggered by a bacterial second messenger.</a> Science. 329 (5993), 845-848 (2010).</li>
<li>Wickiser, J. K., Cheah, M. T., Breaker, R. R., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+kinetics+of+ligand+binding+by+an+adenine-sensing+riboswitch%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">The kinetics of ligand binding by an adenine-sensing riboswitch.</a> Biochemistry. 44 (40), 13404-13414 (2005).</li>
<li>Jucker, F. M., Phillips, R. M., McCallum, S. A., Pardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+a+heterogeneous+free+state+in+the+formation+of+a+specific+RNA-theophylline+complex%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex.</a> Biochemistry. 42 (9), 2560-2567 (2003).</li>
<li>Zhao, Q., Lv, Q., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aptamer+fluorescence+anisotropy+sensors+for+adenosine+triphosphate+by+comprehensive+screening+tetramethylrhodamine+labeled+nucleotides%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.</a> Biosens Bioelectron. 70, 188-193 (2015).</li>
<li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+fluorescence+anisotropy+method+for+detection+of+lead+%28II%29+ion+by+a+G-quadruplex-inducible+DNA+aptamer%5BTitle%5D)+AND+%22Anal+Chim+Acta%22%5BJournal%5D)">A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer.</a> Anal Chim Acta. 812, 161-167 (2014).</li>
<li>Elenko, M. P., Szostak, J. W., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+imaging+of+an+in+vitro-evolved+RNA+aptamer+reveals+homogeneous+ligand+binding+kinetics%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Single-molecule imaging of an in vitro-evolved RNA aptamer reveals homogeneous ligand binding kinetics.</a> J Am Chem Soc. 131 (29), 9866-9867 (2009).</li>
<li>Elenko, M. P., Szostak, J. W., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+binding+experiments+on+long+time+scales%5BTitle%5D)+AND+%22Rev+Sci+Instrum%22%5BJournal%5D)">Single-molecule binding experiments on long time scales.</a> Rev Sci Instrum. 81 (8), 083705(2010).</li>
<li>Zichel, R., Chearwae, W., Pandey, G. S., Golding, B., Sauna, Z. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aptamers+as+a+sensitive+tool+to+detect+subtle+modifications+in+therapeutic+proteins%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.</a> PLoS One. 7 (2), 31948(2012).</li>
<li>Baaske, P., Wienken, C. J., Reineck, P., Duhr, S., Braun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+thermophoresis+for+quantifying+the+buffer+dependence+of+aptamer+binding%5BTitle%5D)+AND+%22Angew+Chem+Int+Ed+Engl%22%5BJournal%5D)">Optical thermophoresis for quantifying the buffer dependence of aptamer binding.</a> Angew Chem Int Ed Engl. 49 (12), 2238-2241 (2010).</li>
<li>Entzian, C., Schubert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+small+molecule-aptamer+interactions+using+MicroScale+Thermophoresis+%28MST%29%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Studying small molecule-aptamer interactions using MicroScale Thermophoresis (MST).</a> Methods. 97, 27-34 (2016).</li>
<li>Valenzano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+and+Identification+of+DNA+Aptamers+to+Tyramine+Using+in+Vitro+Selection+and+High-Throughput+Sequencing%5BTitle%5D)+AND+%22ACS+Comb+Sci%22%5BJournal%5D)">Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing.</a> ACS Comb Sci. 18 (6), 302-313 (2016).</li>
<li>Jauset Rubio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Conglutin+dual+aptamers+binding+distinct+aptatopes%5BTitle%5D)+AND+%22Anal+Bioanal+Chem%22%5BJournal%5D)">beta-Conglutin dual aptamers binding distinct aptatopes.</a> Anal Bioanal Chem. 408 (3), 875-884 (2016).</li>
<li>Breitsprecher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aptamer+Binding+Studies+Using+MicroScale+Thermophoresis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Aptamer Binding Studies Using MicroScale Thermophoresis.</a> Methods Mol Biol. 1380, 99-111 (2016).</li>
<li>Stoltenburg, R., Schubert, T., Strehlitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Selection+and+Interaction+Studies+of+a+DNA+Aptamer+Targeting+Protein+A%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.</a> PLoS One. 10 (7), 0134403(2015).</li>
<li>Kinghorn, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aptamer+Affinity+Maturation+by+Resampling+and+Microarray+Selection%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Aptamer Affinity Maturation by Resampling and Microarray Selection.</a> Anal Chem. 88 (14), 6981-6985 (2016).</li>
<li>Duhr, S., Braun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+molecules+move+along+a+temperature+gradient%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Why molecules move along a temperature gradient.</a> Proc Natl Acad Sci U S A. 103 (52), 19678-19682 (2006).</li>
<li>Braun, D., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trapping+of+DNA+by+thermophoretic+depletion+and+convection%5BTitle%5D)+AND+%22Phys+Rev+Lett%22%5BJournal%5D)">Trapping of DNA by thermophoretic depletion and convection.</a> Phys Rev Lett. 89 (18), 188103(2002).</li>
<li>Duhr, S., Arduini, S., Braun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermophoresis+of+DNA+determined+by+microfluidic+fluorescence%5BTitle%5D)+AND+%22Eur+Phys+J+E+Soft+Matter%22%5BJournal%5D)">Thermophoresis of DNA determined by microfluidic fluorescence.</a> Eur Phys J E Soft Matter. 15 (3), 277-286 (2004).</li>
<li>Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+interaction+studies+using+microscale+thermophoresis%5BTitle%5D)+AND+%22Assay+Drug+Dev+Technol%22%5BJournal%5D)">Molecular interaction studies using microscale thermophoresis.</a> Assay Drug Dev Technol. 9 (4), 342-353 (2011).</li>
<li>He, K., Dragnea, V., Bauer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenylate+Charge+Regulates+Sensor+Kinase+CheS3+To+Control+Cyst+Formation+in+Rhodospirillum+centenum%5BTitle%5D)+AND+%22MBio%22%5BJournal%5D)">Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum.</a> MBio. 6 (3), 00546(2015).</li>
<li>Brvar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-based+discovery+of+substituted+4%2C5%27-bithiazoles+as+novel+DNA+gyrase+inhibitors%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">Structure-based discovery of substituted 4,5'-bithiazoles as novel DNA gyrase inhibitors.</a> J Med Chem. 55 (14), 6413-6426 (2012).</li>
<li>Pogorelcnik, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4%2C6-Substituted-1%2C3%2C5-triazin-2%281H%29-ones+as+monocyclic+catalytic+inhibitors+of+human+DNA+topoisomerase+IIalpha+targeting+the+ATP+binding+site%5BTitle%5D)+AND+%22Bioorg+Med+Chem%22%5BJournal%5D)">4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIalpha targeting the ATP binding site.</a> Bioorg Med Chem. 23 (15), 4218-4229 (2015).</li>
<li>Jhaveri, S., Rajendran, M., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+selection+of+signaling+aptamers%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">In vitro selection of signaling aptamers.</a> Nat Biotechnol. 18 (12), 1293-1297 (2000).</li>
<li>Khavrutskii, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+purification-free+method+of+binding+affinity+determination+by+microscale+thermophoresis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Protein purification-free method of binding affinity determination by microscale thermophoresis.</a> J Vis Exp. (78), (2013).</li>
<li>Ramakrishnan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+cocaine-antibody+interactions+in+buffer+and+human+serum%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Probing cocaine-antibody interactions in buffer and human serum.</a> PLoS One. 7 (7), 40518(2012).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiviral+activity+and+interaction+mechanisms+study+of+novel+glucopyranoside+derivatives%5BTitle%5D)+AND+%22Bioorg+Med+Chem+Lett%22%5BJournal%5D)">Antiviral activity and interaction mechanisms study of novel glucopyranoside derivatives.</a> Bioorg Med Chem Lett. 25 (18), 3840-3844 (2015).</li>
<li>Wan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+the+molecular+recognition+of+the+granuphilin+C2A+domain+with+PI%284%2C5%29P2%5BTitle%5D)+AND+%22Chem+Phys+Lipids%22%5BJournal%5D)">Insights into the molecular recognition of the granuphilin C2A domain with PI(4,5)P2.</a> Chem Phys Lipids. 186 (4,5), 61-67 (2015).</li>
<li>Harazi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Interaction+of+UDP-N-Acetylglucosamine+2-Epimerase%2FN-Acetylmannosamine+Kinase+%28GNE%29+and+Alpha-Actinin+2+Is+Altered+in+GNE+Myopathy+M743T+Mutant%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.</a> Mol Neurobiol. , (2016).</li>
</ol>

CE und TS sind Mitarbeiter von 2bind GmbH, die biophysikalischen analytische Dienste zur Verfügung stellt. Publikationsgebühren für dieses Video-Artikel sind für die von 2bind GmbH bezahlt.

Qualitätskontrollen: Unspezifische Klebe- / Adsorption von Probenmaterial an Oberflächen sowie Aggregationseffekte, haben einen dramatischen Einfluss auf die Qualität der Affinitätsdaten. Doch nur wenige state-of-the-art Technologien bieten genaue und schnelle Möglichkeiten zu überwachen und diese Effekte zu vermeiden. MST bietet integrierte Qualitätskontrollen, die erkennen und helfen, diese Probleme zu überwinden, so dass für die schrittweise Optimierung der technischen Einrichtung. ...

Wechselwirkung zwischen Molekülen ist die Basis der Natur. Daher Wissenschaftler in vielen Bereichen der Grundlagen- und angewandten Forschung versuchen die grundlegenden Prinzipien der molekularen Wechselwirkungen verschiedener Arten zu verstehen. Micro Thermophorese (MST) können Wissenschaftler die schnelle, präzise und kosteneffiziente und qualitätskontrollierte Charakterisierung von molekularen Wechselwirkungen in Lösung durchzuführen, mit einer freien Wahl von Puffern. Es gibt bereits mehr als 1.000 Publikationen MST mit, ab 2016 allein, beschreibt verschiedene Arten von Analysen, einschließlich Bibliothek Vorführungen, Ereignis Validierungen Bindung, kompetitive Assays, und Experimente mit mehreren Bindungspartnern 1-8. Im Allgemeinen erlaubt MST die Untersuchung der klassischen Bindungsparameter wie Bindungsaffinität (pM bis Mm), Stöchiometrie und Thermodynamik, der jede Art von molekularen Wechselwirkungen. Ein großer Vorteil der MST ist die Fähigkeit, Bindungsereignisse unabhängig von der Größe der Interaktionspartner zu studieren. Auch Heraus-fordernd Wechselwirkungen zwischen kleinen Nukleinsäure-Aptamere (15-30 nt) und Ziele wie kleine Moleküle, Medikamente, Antibiotika oder Metaboliten quantifiziert werden können. Aktuelle state-of-the-art Technologien Aptamer-Target - Wechselwirkungen sind entweder im Labor intensiv und sehr komplex zu charakterisieren oder scheitern Aptamer-kleines Molekül 9,10 Wechselwirkungen zu quantifizieren. Oberflächenplasmonresonanz (SPR) -basierte Assays 11,12 und wirklich markierungsfreie kalorimetrischen Ansätze wie Isothermen Titrations - Kalorimetrie (ITC) 13-15, isokratische Elution 16, Gleichgewicht Infiltrations 17,18, in-line Sondieren 19, Gel- Shift - Assays, Stopped fl ow Fluoreszenz- Spektroskopie 20,21, Fluoreszenzanisotropiemessungen (FA) 22,23, Einzelmolekül - Fluoreszenz - Bildgebung 24,25 und Bio-Schicht - Interferometrie (BLI) 26 sind ebenfalls entweder ungenau oder nicht kompatibel mit Aptamer-kleines Molekül Wechselwirkungen. Andere principal Probleme dieser Verfahren sind eine geringe Empfindlichkeit, hohe Probenverbrauch, Immobilisation, Massentransportbeschränkungen auf Oberflächen und / oder Puffer Einschränkungen. Nur wenige dieser Technologien bieten integrierte Kontrollen für die Aggregation und Adsorption Effekte. MST stellt ein leistungsfähiges Werkzeug für Wissenschaftler , diese Einschränkung zu überwinden , um die Wechselwirkungen zwischen Aptamere und kleine Moleküle , 27-29, sowie andere Ziele, wie Proteine 30-33 zu studieren. Die Technologie beruht auf der Bewegung von Molekülen durch Temperaturgradienten. Diese gerichtete Bewegung, die so genannte &quot;Thermophorese,&quot; hängt von der Größe, der Ladung und Hydrathülle des Moleküls 34,35. Die Bindung eines Liganden an das Molekül direkt an mindestens einen dieser Parameter zu verändern, in einer veränderten thermophoretische Mobilität führt. Liganden mit kleinen Größen haben nicht erhebliche Auswirkungen in Bezug auf die Größenänderung von der ungebundenen zum gebundenen Zustand, aber sie können dr haben<html> amatic Auswirkungen auf die Hydrathülle und / oder Ladung. Die Veränderungen in der thermophoretische Bewegung der Moleküle nach der Wechselwirkung mit dem Bindungspartner ermöglicht die Quantifizierung der Grundbindungsparameter 2,7,34,36,37. Wie in 1A dargestellt ist , besteht der MST Vorrichtung eines Infrarotlasers auf die Probe innerhalb der Glaskapillaren fokussiert die gleiche Optik wie für die Fluoreszenzdetektion. Die thermophoretischer Bewegung von Proteinen über die Eigenfluoreszenz von Tryptophane 6 oder eines fluoreszierend 3,8 Interaktionspartner markiert überwacht werden kann , während der Laser einen Temperaturgradienten (AT von 2-6 ° C) herstellt. Die sich ergebende Temperaturdifferenz im Raum, &amp; Delta; T, führt zur Verarmung oder Anreicherung von Molekülen im Bereich der erhöhten Temperatur, die durch die Soret quantifiziert werden kann Koeffizienten (S T): g &quot;/&gt; c hot stellt die Konzentration in dem erwärmten Bereich, und c kalt ist die Konzentration in dem anfänglichen Kaltbereich. Wie in 1B gezeigt , um eine typische MST Versuchsergebnisse in einer MST Bewegungsprofil (Zeitverlauf), die aus verschiedenen Phasen, die durch ihre jeweiligen Zeitrahmen getrennt werden können. Die anfängliche Fluoreszenz wird in den ersten 5 s in Abwesenheit des Temperaturgradienten gemessen, um die genaue Start Fluoreszenz zu definieren und für Photobleaching oder photoenhancement zu überprüfen. Der Temperatursprung (T-Jump) repräsentiert die Phase, in der die Fluoreszenzänderungen vor thermophoretischer Bewegung. Diese anfängliche Abnahme der Fluoreszenz hängt von wärmeabhängige Veränderungen von fl uorophore Quantenausbeute. Die Thermophorese Phase folgt, in der die Fluoreszenz abnimmt (oder zunimmt) aufgrund der thermophoretische Bewegung der Moleküle, bis die Stationärzustandsverteilung erreicht wird.Die umgekehrte TJump und die gleichzeitige Rückdiffusion der fluoreszierenden Moleküle können wie in 1B angegeben beobachtet werden , nachdem der Laser ausgeschaltet ist. Um grundlegende Bindungsparameter, verschiedene Molverhältnisse der Interaktionspartner für den Zugriff werden analysiert und verglichen. Typischerweise werden 16 unterschiedliche Verhältnisse in einem MST Experiment untersucht, wohingegen der optische sichtbaren Molekül konstant gehalten wird und mit zunehmender Menge des unmarkierten Liganden zugeführt wird. Die Wechselwirkung zwischen den beiden Bindungspartner induziert Veränderungen in der Thermophorese, und somit in der normalisierten Fluoreszenz, F Norm, die wie folgt berechnet wird: <img alt="Gleichung" src="/files/ftp_upload/55070/55070eq2.jpg" /> F heiß und F kalt stellen Intensitäten fluoreszenz bei de fi nierten Zeitpunkte der MST Spuren gemittelt. Bindungsaffinitäten (K d oder EC 50 -Werte) können durch curv berechnet werdene Fitting (1C). Insgesamt ist MST ein leistungsfähiges Werkzeug molekularen Wechselwirkungen jeglicher Art zu studieren. Dieses Manuskript bietet ein Protokoll das herausfordernde Interaktion zwischen dem kleinen Molekül Adenosintriphosphat (ATP; 0,5 kDa) zu charakterisieren und die 25-nt kurze ssDNA Aptamer DH25.42 (7,9 kDa). Im Laufe des Manuskripts wird die Bindungsstelle des Aptamers an das ATP-Molekül an die Adenin-Gruppe des ATP kartiert unten.

<table><tbody><tr><td>Aptamer binding buffer</td><td></td><td></td><td>20 mM Tris pH 7.6; 300 mM NaCl; 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;; 0.01% Tween-20</td></tr><tr><td>Fluorescently labeled ATP aptamer</td><td>IDT, Leuven, Belgium</td><td></td><td>sequence: DH25.42 50-Cy5-CCTGGGGGAGT-&lt;br/&gt;ATTGCGGAGGAAGG-3</td></tr><tr><td>ATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2383</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>ADP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2754</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>AMP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A2252</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Adenine</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A8626</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>SAM</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>A7007</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>dATP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>11934511001</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>CTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>C1506</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>GTP</td><td>Sigma Aldrich, Germany&amp;nbsp;</td><td>G8877</td><td>10 mM stock solutions stored at - 20 &amp;deg;C</td></tr><tr><td>Monolith NT.115&amp;nbsp;</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-G008</td><td>Blue/Red Channel&lt;br/&gt; MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector</td></tr><tr><td>Monolith NT.115 capillaries Standard</td><td>NanoTemper Technologies, Munich, Germany</td><td>MO-K002</td><td></td></tr><tr><td>Eppendorf PCR tubes</td><td>Eppendorf, Germany</td><td>30124537</td><td></td></tr><tr><td>Monolith control software. 2.1.33, pre-installed on the device</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>MO.affinity analysis v2.1.1</td><td>NanoTemper Technologies, Munich, Germany</td><td></td><td></td></tr><tr><td>Kaleidagraph 4.5.2</td><td>Synergy Software</td><td></td><td></td></tr></tbody></table>

mapping the binding site of an aptamer on atp using microscale thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

In dieser Studie wurde MST die Bindungsstelle des DNA - Aptamer DH25.42 18 an ATP zu charakterisieren aufgetragen. Im Gegensatz zu anderen Studien , die die Wechselwirkung von ATP oder ATP-nachahmt kleine Moleküle mit Proteinen zufällig markiert mit einem oder mehreren Fluorophoren 38-40 charakterisierenden umfasst diese Studie eine markierte Version des 7,9 kDa ssDNA Aptamer mit einem Cy5 - Molekül am 5&#39;Ende . Verschiedenen ATP-Derivate und verwandte Molekü...

Watch this Scientific Journal Video about Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis at JoVE.com

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Kartierung der Bindungsstelle eines Aptamer auf ATP Mit Micro Thermophorese

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

mapping-binding-site-an-aptamer-on-atp-using-microscale

Research

JoVE Journal

Biochemistry

10.6K Aufrufe.  2bind GmbH. MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Serie: Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Phthalsäure Acid Ester-Bindung DNA-Aptamer Auswahl, Charakterisierung und Anwendung eines elektrochemischen Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

Forschung

Chemie

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Einsatz der mikroskaligen Thermophorese zur Messung von Protein-Lipid-Wechselwirkungen

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Biochemie

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Messung der Nukleotidbindung an intakte, funktionelle Membranproteine in Echtzeit

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

Ein auf Durchflusszytometrie basierender Zelloberflächenproteinbindungsassay zur Beurteilung der Selektivität und Spezifität eines Antikrebs-Aptamers

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Krebsforschung

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Primer-Free Aptamer Selection mit einem zufälligen DNA-Bibliothek

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biologie

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

Bestimmung der thermodynamischen und kinetischen Assoziation eines DNA-Aptamers und Tetracyclins mittels isothermer Titrationskalorimetrie

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

Aptamer-basierte Target-Detektion durch eine 3-stufige isotherme G-Quadruplex-Amplifikationsreaktion

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

Biotechnik

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification-freie Methode der Bindungsaffinität Bestimmung durch Microscale Thermophorese

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Source: Mittermaier, A. K., et al. <a href="https://app.jove.com/v/55959">Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions.</a> J. Vis. Exp. (2017)
This video demonstrates differential scanning calorimetry &#8212; a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic analysis.

Dynamische Differenzkalorimetrie zur Untersuchung der Interaktion zwischen DNA-Aptamer und thermolabilen Liganden

Source: Mittermaier, A. K., et al. Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions. ...